Stroke is one of the leading causes of death worldwide and a major cause of long-term disability. Although many clinical trials have targeted stroke patients, only thrombolysis has so far emerged as an effective treatment. Nonetheless, new therapeutic targets have emerged. Calcium dysregulation has long been implicated in brain injury in the setting of stroke. Our prior work on this grant, as well as work from others, has shown that the heat shock protein 70 kDa (HSP70) family of chaperones or stress proteins, even if expressed after ischemia, can protect the brain. The regulation of apoptosis by the BCL2 family of proteins is another important determinant of ischemic outcome. These pathways converge to control cellular calcium homeostasis and both apoptotic and necrotic cell death. The endoplasmic reticulum (ER) and mitochondria interact at specific sites called the mitochondrial associated ER membrane (MAM) to regulate cellular calcium homeostasis and cell death. MAM is a critical site of stress/chaperone protein and BCL2 family interaction, and is central to determining the outcome from cerebral ischemia. We recently found that overexpressing GRP78, an ER chaperone and HSP70 family member, preserves mitochondrial function, reduces mitochondrial Ca2+ overload, and improves brain cell survival after stress. We also found that GRP78 translocates to the mitochondrial inner membrane after stress in astrocytes, CNS glial cells that are known to be integral to neuronal survival. In Aim 1 of this proposal we will investigate the role of translocated GRP78 in mitochondrial function by comparing wild type and mitochondrially targeted GRP78. The discovery of posttranscriptional gene silencing by miRNAs has led to an explosion of new hypotheses in human disease. Studies of miRNAs in cerebral ischemia are recent, and most have focused on profiling changes in miRNAs. We recently reported that reducing or blocking miR-181, a brain-enriched miRNA, protects the brain from stroke in the initial post-injury period. Aim 2 is a translational aim and ill follow up these studies to determine the effects of altering miR-181 on long term behavioral outcome from stroke, test post-treatment, and determine whether miR-181 is also effective in female animals. We recently demonstrated that miR-181 can target both GRP78 and anti-apoptotic BCL2 family members BCL2 and MCL1. Therefore, the role of miR-181 in ER-mitochondrial calcium transfer in MAM will be studied. Using computational miRNA target prediction we identified miR-200 as potentially targeting GRP75, a mitochondrial chaperone, and BCL2. Aim 3 of this proposal will focus on the mechanism of protection by reducing miR-181 levels investigating in detail effects on ER and mitochondrial Ca2+, and mitochondrial function. Overall this proposal exams a novel hypothesis: that miRNAs act as master regulators of chaperones and BCL2 family members influencing Ca2+ homeostasis, mitochondria-ER crosstalk, and outcome after cerebral ischemia.